Downregulated lignin transgenic black cottonwood (Populus trichocarpa) was used to elucidate the effect of lignin and xylan content on enzymatic saccharification. The lignin contents of three transgenic samples (4CL1-1, 4CL1-4, and CH8-1-4) were 19.3, 16.7, and 15.0 %, respectively, as compared with the wild type (21.3 %). The four pretreatments were dilute acid (0.1 % sulfuric acid, 185 °C, 30 min), green liquor (6 % total titratable alkali, 25 % sulfidity based on TTA, 185 °C, and 15 min.), autohydrolysis (185 °C, 30 min), and ozone delignification (25 °C, 30 min). Following the pretreatment, enzymatic saccharification was carried out using an enzyme charge of 5 FPU/g of substrates. The removal of lignin and hemicellulose varies with both the types of pretreatments and the lignin content of the transgenic trees. Due to the greatest removal of lignin, green liquor induced the highest sugar production and saccharification efficiency, followed by acid, ozone, and autohydrolysis in descending order. The results indicated that lignin is the main recalcitrance of biomass degradation. At a given lignin content, pretreatment with ozone delignification had lower saccharification efficiency than the other pretreatment methods due to higher xylan content.